Succinic acid has drawn much interest because it has been used as a precursor of numerous chemicals including pharmaceuticals and biodegradable polymers. Succinic acid is a member of the C4-dicarboxylic acid family and it is commercially manufactured by hydrogenation of maleic anhydride to succinic anhydride, followed by hydration to succinic acid. Recently major efforts have been made to produce succinic acid by microbial fermentation using a renewable feedstock. Many attempts have been made to metabolically engineer the anaerobic central metabolic pathway of Escherichia coli (E. coli) to increase succinate yield and productivity. E. coli is extensively used in industry as a host for many products due to the ease of genetic manipulation coupled to its fast growth rate, standardized cultivation techniques and cheap media. It is for this reason and for the need to produce succinic acid economically at high concentrations and yields that E. coli has been considered as a potential candidate to produce this product of industrial interest.
It is well known that under anaerobic conditions and in the absence of exogenous electron acceptors, E. coli metabolizes glucose to a mixture of fermentative products consisting primarily of acetate, ethanol, lactate and formate with smaller quantities of succinate. NADH produced by the catabolism of glucose is regenerated to NAD+ through the reduction of intermediate metabolites derived from glucose in order to continue with glycolysis. The distribution of products varies according to the strain and growth conditions and is dictated by the way reducing equivalents generated in the form of NADH are consumed so that an appropriate redox balance is achieved by the cell.
Numerous efforts have been undertaken to make succinate the major fermentation product in E. coli. Some genetic manipulations previously studied are: deletion of the fermentative lactate dehydrogenase (LDH) pathway, deletion of both the LDH and pyruvate formate lyase (PFL) pathways and deletion of multiple pathways including PFL and LDH pathways with an additional ptsG mutation which restored the ability of the strain to grow fermentatively on glucose and resulted in increased production of succinic acid. Other studies include overexpression of phosphoenolpyruvate carboxylase, (PEPC), overexpression of the malic enzyme and overexpression of pyruvate carboxylase (PYC). Besides these genetic manipulations, external means have been developed in order to increase succinate production such as utilizing a dual phase fermentation production mode which comprises an initial aerobic growth phase followed by an anaerobic production phase, or by changing the headspace conditions of the anaerobic fermentation using carbon dioxide or hydrogen. It has been suggested that an external supply of H2 might serve as a potential electron donor for the formation of succinic acid, a highly reduced fermentation product when compared to glucose.
Under fully anaerobic conditions, the maximum theoretical yield (molar basis) of succinate from glucose is one based on the number of reducing equivalents provided by this substrate. One mole of glucose can provide only two moles of NADH, and two moles of NADH can only produce one mole of succinate, therefore, in order to surpass the maximum theoretical yield it is necessary to use part of the carbon coming from glucose to provide additional reducing power to the system.
Metabolic engineering has the potential to considerably improve process productivity by manipulating the throughput of metabolic pathways. Specifically, manipulating intermediate substrate levels can result in greater than theoretical yields of a desired product.